Increased DNA binding specificity for antitumor ecteinascidin 743 through protein-DNA interactions?

Ecteinascidin 743 (ET743) is a marine natural product consisting of three linked tetrahydroisoquinoline subunits and an active carbinolamine functional group (Figure 1). ET743 displays potent antiproliferative activity against a variety of tumor cells and is currently undergoing phase II clinical trials.1 ET743 is known to bind to the minor groove of several DNA triplets containing a central guanine (e.g. AGC, CGG, TGG)2 and to alkylate the exocyclic amino group of this purine base,3 but the mechanism by which it exerts its anticancer activity remains to be elucidated. We have recently studied, by molecular dynamics simulations in aqueous solution, the complexes of ET743 with two DNA target sequences and shown that drug binding brings about widening of the minor groove and bending toward the major groove.4 The degree of bending was found to be in very good agreement with results from gel electrophoresis experiments,5 and the major local bending element identified was an increase in roll at the base pair step involved in covalent bond formation. Prevailing ideas about the mode of action of ET743 are that (i) by inducing bends in DNA, the drug could be serving a surrogate protein function by juxtaposing specified DNA fragments not contiguous in the primary sequence, (ii) one or more proteins could be recruited to the drug-DNA complex, and/or (iii) drug binding could disrupt the interaction of DNA-binding proteins with their target sites.6 Indeed, it has been shown that binding of transcription factor NF-Y is impaired7 and topoisomerase I is poisoned8 by ET743, albeit in both cases at concentrations much greater than are necessary for achieving its cytotoxic effect. On the other hand, at pharmacologically relevant concentrations, ET743 is also known to decrease induced activation of expression of a number of genes,9 the best studied of which is MDR1,9,10 the gene whose overexpression is responsible for multidrug resistance in cancer cells. The MDR1 gene is regulated by NF-Y, which binds to a CCAAT box,11 and also by Sp1, which binds to a GC box.12 CCAAT contains a TGG site on the complementary strand, which certainly is one of the preferred binding sequences for this drug.2,3 When looking for other possible binding sites on the 5′-flanking promoter region of MDR1 (Figure 2),13 we focused on the 5′-CGGGCCGGG-3′ sequence to which Sp1 binds. This GC box contains two possible CGG binding sites on one strand and an alternative CGG binding site on the complementary strand. Sp1 is predicted to contain three contiguous Cys2-His2 zinc finger domains14 near the C-terminus which are highly homologous15 to other zinc fingers whose structures have been solved, both free and complexed to their cognate DNA sequences. Particularly noteworthy is the transcriptional regulator EGR-1 (Zif268)16 which binds to the DNA sequence 5′CGCCCCCGC-3′ (EGR site) and variants of which have been cocrystallized with a number of different oligonucleotides.17 From this and related work,18 it has been learned that each Cys2-His2 zinc finger is composed of two short â-strands followed by an R-helix19 and makes its primary contacts with the DNA major groove in a three-base pair subsite. In doing so they cause a distinctive distortion in the DNA molecule.20 Unexpectedly, we have found that the minor grooves of DNA when bound to the zinc fingers of EGR-1 and in the covalent complex with ET743 are virtually superimposable (Figure 3). This striking similarity raises the interesting possibility that ET743 preferentially targets the minor groove of DNA when bound to a zinc fingercontaining transcription factor, very likely but probably not exclusively Sp1.14b In fact, molecular modeling of the Sp1-DNA-ET743 complex is relatively straightforward (Figure 3B) as the widened minor groove in the homology-based zinc finger region of the Sp1-DNA binary complex21 and the more readily accessible 2-amino group of guanine provide a binding site for ET743 that is highly preshaped for fitting. Since no additional distortions have to be inflicted by the drug upon binding,4 we propose that this prestructured site could provide an important additional element of selectivity so as to be preferred over any of the vast number of suitable DNA triplets present in the entire genome which are presumably found in a more or less standard B-DNA conformation. Thus, ET743 binding to DNA would depend not only on the well-defined hydrogenbonding rules necessary for sequence recognition and adduct formation2,3 but also on the protein-induced preorganization of a DNA stretch that becomes structurally complementary to the wedge shape of the drug molecule.3,4 The functional groups of ET743 and related analogues22 not involved in DNA binding that protrude out of the minor groove and are known to be essential for activity are likely to interact with other protein residues. Their role could be either stabilizing the ternary complex, thereby preventing dissociation of Sp1, or else promoting a conformational change in a spatially con* To whom correspondence should be addressed. Tel: +34 918 854 514. Fax: +34 918 854 591. E-mail: [email protected]. † Universidad de Alcalá. ‡ Pharma Mar. Figure 1. Chemical structure of ecteinascidin 743 (ET743). 4367 J. Med. Chem. 2000, 43, 4367-4369